1. Field of the Invention
The invention relates to microorganisms as carriers of heterogeneous nucleotide sequences coding for antigens and protein toxins, a process of manufacturing thereof as well as corresponding plasmids or expression vectors. These microorganisms can be used as medicaments, in particular as tumor vaccines for the treatment of various tumors.
2. Description of the Related Art
Immunotherapy of cancer represents a promising option of tumor-treatment. A multiplicity of clinical trials using different approaches concentrates on its efficiency in patients. In principle, a distinction is drawn between passive and active immunotherapy.
Active immunotherapy aims at the induction of a vaccine-related tumor-specific immune response. The latter is currently being clinically probed using several different approaches. For instance there are so called whole-cell vaccines, whose feed stock are tumor cells that are either directly obtained from the patient (autologous) or derived from appropriate cell lines (heterologous). These cells are then usually inactivated, differentially manipulated and (re)applied to the patient.
In contrast, antigen-specific vaccines contain one (or more) tumor-specific antigens, parts of the antigen or the specific antigen-coding DNA as well as so called anti-idiotype vaccines. Normally these vaccines are not isolated, but injected in combination with an appropriate carrier. Hence, on one hand different classic adjuvants are utilized, but likewise combinations with biological immuno-stimulants such as cytokines.
For the purpose of immuno-stimulation, approaches are being applied, that contain antigen associated with immuno-stimulants, such as tetanus toxin. Furthermore, there exist attempts, applying antigens in combination with dendritic cells. And finally there are several attempts with recombinant live-vaccines with viral or bacterial carriers.
Fusion proteins of bacterial toxins, such as tetanus toxin, shiga toxin, lethal toxin or cholera toxin, as adjuvants with an antigen are utilized as vaccines especially against infections for quite some time (Freytag and Clements, 1999). Additionally, native toxins, often merged with a target-cell specific molecule, such as a cell surface molecule of tumor cells, are also used in order to destroy target cells.
At this, fusion proteins with the native toxin, that generally comprise an enzymatic unit and a protein-binding domain, develop their optimum effect in use as adjuvants (Freytag and Clements, 1999). By means of these vaccines a satisfying immune response is obtained even after mucosal and particularly after oral immunization. The trouble with these fusion proteins is, that native toxins are highly toxic and therefore cannot be established in humans (Holmgren et al., 2005).
A whole string of research is thus occupied with detoxification of toxins that at the same time preserve the adjuvant-effect. However, since in most cases the adjuvant-effect coincides with enzymatic-activity, which is responsible for the toxic effect (Lycke et al., 1992), detoxification cannot be performed in a straightforward way, even if it appears to be possible for some toxins that do not lose their enzymatic, adjuvant activity (Hormozi et al., 1999; Lycke et al., 1992).
In the case of cholera toxin (CT) several attempts of detoxification are pursued (Agren et al., 1999; Byun et al., 2001; Eriksson et al., 2004; Kweon et al., 2002; Sanchez et al., 2002), for which, however, the use as mucosal adjuvant prevails (Freytag and Clements). Therefore, above all, efficient induction of an antibody response (primarily mucosal IgA), which receive an increased toxin-related MHC class II restricted T-cell support, is the main prerequisite for a mucosal adjuvant for a vaccine that consists of protein-antigen and a fused or co-applied toxin (Freytag and Clements).
As for cholera toxin, especially its B-subunit (CtxB) is tested as adjuvant since it is responsible for binding to the GM-1 receptor and does not show toxic effects when isolated (Holmgren et al.). Protein fusions with CtxB are characterized by primary induction of so called Th2 immune responses. These are T-cell responses, which are mainly characterized by cytokines, such as IL-4 or IL-6, and which primarily cause induction of antibodies, but which do not at all or at most restrictedly initiate a cellular immune response, particularly of cytotoxic T cells (CTL) (Holmgren et al.).
In addition, CtxB as mucosal adjuvant induces protein-antigen systemic tolerance. Systemic tolerance describes the depletion or inactivation of antigen-specific lymphocytes, in particular T-cells or B-cells. This kind of approach is therefore inapplicable for the induction of a systemic immune response (Holmgren et al.).
In contrast to mucosal application intraperitoneal or subcutaneous application of a toxin-antigen fusion protein is able to induce systemic as well as low cytotoxic responses. This has indeed been utilized for tumor-vaccination in model systems (refer to e.g. (Becerra et al., 2003)). However such response is also obtained with the purified antigen itself and is primarily depending on the adjuvant used. Apart from the fact that the measured CTL responses in the model are rather low, there was no evidence that the protection was even depending on these effects. Moreover, the antigen was not applied orally, but only by direct injection (s.c., i.d., i.m., i.p.) of the antigen.
Antigen proteins fused to a detoxified toxin are generally ineffective if applied as oral tumor vaccine. The main reasons are, if present at all, only low induction of a systemic immune response or even, in the case of CtxB, induction of systemic tolerance as well as induction of mucosally restricted antibody and Th2 type immune responses.
McSorley et al., for instance, showed that nasal immunization with a CtxB-antigen fusion protein (which for a tumor vaccine represents the preferred way of inducing a systemic response) preferably tolerates and therefore inactivates Th1 cells, whereas Th2 cells are not influenced (McSorley et al., 1998). Th2 responses are characterized by T-helper cells that predominantly produce IL-4 or IL-6. These cytokines are particularly responsible for the initiation of antibody production by B-cells, which provide protection in the case of most conventional vaccines. In contrast, Thl T-cells mostly secrete IL-2 and IFN-gamma, thus cytokines, which play a role in cellular immune response. Depending on the objective of an immunization strategy it is of crucial importance, whether an antibody dominated Th2 immune response (so-called Th2 bias) or a cellular dominated Th1 response (so called Th1 bias) is initiated.
By contrast, systemic induction of a Th1 dominated cellular immune response with IFN-gamma producing T-helper cells and induction of cytotoxic T-cells (CTL) is indispensable for tumor immunotherapy.
The prior art shows that toxins can function as adjuvants. Especially cholera toxin (CT) shows a strong adjuvant effect. However, this effect is significantly attenuated as soon as the toxin is detoxified. Regarding its subunit CtxB, oral application even induces systemic tolerance. This problem can be partially avoided by nasal application. However, if nasal administration is applied, other problems arise, in particular such as the ones associated with CtxB subunit concerning the expression of GM-1 receptor in the brain (van Ginkel et al., 2000), which are reflected in an increasing risk of particular severe side effects (Mutsch et al., 2004).
To overcome these problems, it may be possible to generate recombinant live-vaccines that co-express toxins and (heterologous) antigens. This option has already been pursued with regard to infection vaccines, i.e. vaccines that are directed against a specific pathogen, for instance the tuberculosis pathogen, and are meant to induce immunity against that pathogen.
In the course of developing such infection vaccines, recombinant toxins fused to appropriate (heterologous) antigens have already been generated, using several recombinant bacterial strains that are administered orally, either as alive or inactivated vaccine. In most cases, the generated strains only expressed a recombinant toxin. Hence, these approaches primarily aimed at solely immunizing against the toxin itself (induction of an antibody response) and, consequently, the toxin expressing strain (pathogen). These kinds of vaccines are not suitable for use as potential tumor vaccines in tumor therapy and, not surprisingly, corresponding trials do not mention any such possible application (Reveneau et al., 2002) (Vertiev et al., 2001) (Freytag and Clements, 1999; Jackson et al., 1996).
With respect to such infection vaccines, in the course of induction of an antibody response the toxin does not function as adjuvant, it is the induction of a mucosal antibody immune response which prevails. These infection vaccine trials include no or only a very weak systemic immune response. In contrast to tumor vaccines, such vaccines do not pursue an induction of cellular immune response, especially of cytotoxic T-cells. The toxin expressing bacterial pathogens normally show an extracellular live pattern and therefore do not contribute to an activation of CTL, i.e. the protection conferred by such infection vaccines is CTL independent.
The following bacterial strains have been used in the mentioned infection vaccine trials: recombinant Lactobacilli (Reveneau et al., 2002), Listeria (Vertiev et al., 2001), Bacillus anthracis (Mendelson et al., 2005; Mesnage et al., 1999), Shigellae (Anderson et al., 2000; Tzschaschel et al., 1996b), E. coli (Walker et al., 1992), Vibrio (Butterton et al., 1995; Chen et al., 1998; Thungapathra et al., 1999) and Salmonella (Jackson et al., 1996).
In addition, it was tried to enhance mucosal immune responses by surface presentation (Konieczny et al., 2000) or secretion of the toxin as heterologous antigen (Salmonella, Shigellae: (Garmory et al., 2003; Orr et al., 1999; Su et al., 1992; Tzschaschel et al., 1996a; Tzschaschel et al., 1996b), Yersinia: (Sory and Cornelis, 1990), Vibrio (Ryan et al., 1997a), E. coli: (Zhu et al., 2006)). However, in these additional cases the toxins themselves represent the antigens to which the immune response is directed to.
Thus, it has to be noted that the toxins neither function as adjuvants nor did the describing studies intend to express toxins as fusion proteins with an additional separate (heterologous) antigen. Moreover, fusions proteins with CT or CtxB were neither generated with these systems nor were they suggested.
Consequently, fusion proteins mentioned in above cited documents are fusion proteins of a peptidic secretion signal, such as HlyA, and a toxin protein, but not fusion proteins, consisting of a toxin and a (heterologous) antigen.
The primary objective of these studies was to merely obtain an optimum mucosal immune response (Tzschaschel et al., 1996a). Induction of a systemic immune response was not pursued. Yet if measured at all, analyses of systemic immune responses were restricted to antibodies (particularly systemic IgA) only, since protection in these kind of models is mainly mediated by antibodies (compare e.g. [31]). Induction of a systemic cellular immune response, in particular a cytotoxic T-cell response, is not described. The fusion with a secretion signal was primarily used in order to increase the solubility of the toxins and enhance their stability, respectively, since a strong, mere cytoplasmatic expression often results in the production of insoluble aggregates (Gentschev et al., 2002a).
In this respect, some authors observe that fusion proteins with a secretion signal cause a fast cytoplasmatic degradation (Tzschaschel et al., 1996a), whereas others observe a stabilization (Orr et al., 1999). The prior art, hence, is contradictory here. So far, previous experiments with secreted toxins (toxin+secretion signal) are above all aimed at increasing stability, which obviously was not achieved in all cases.
One reason certainly is found in variable expression intensity and plasmid stability. Tzschaschel et al. describe, that the plasmid system used, is highly instable and above all, without selection, is merely found in a few bacteria. As a possible solution the authors use chromosomal integration via a mini-transposon (Tzschaschel et al., 1996a; Tzschaschel et al., 1996b).
However, such a system possesses several disadvantages. On one hand the point of integration is not defined, which can lead to an undesired phenotype alteration of the host strain (e.g. increased/decreased expression of flanking genes). On the other hand the expected expression level using a single genomic copy is only low, which has a negative effect on immunogenicity. After all, chromosomal transposon integration is relatively instable, for it leads quite frequently to spontaneous excisions at the sides of the repetitive elements.
As mentioned above, the prior art is contradictory concerning the stability of a secreted heterologous toxin.
Garmory et al. even assume, that secretion of a heterologous antigen does not have any special benefit with regard to immunogenicity (Garmory et al., 2002; Roland et al., 2005). In other cases increased systemic antibody response to a secreted toxin after intravenous administration has indeed been observed, but not after oral application. All the contrary, oral application is even indirectly put into question (Roland et al., 2005).
Eventually, studies with a gram-positive strain (Lactobacillus plantarum) which produces tetanus toxin showed no significant difference in induction of a systemic antibody response compared to strains that secrete the toxin, present it bound to their membrane or contain the toxin cytoplasmatically (Reveneau et al., 2002).
Systemic cellular immune responses, in particular cytotoxic T-cell responses, and the resulting protection, were not studied nor described.
The above cited prior art of toxin-based infection vaccines thus cannot declare if a secretion of toxin used as adjuvant represents an advantage, concerning the induction of a systemic (cellular) immune response. All the contrary, above cited studies rather point the stability problem out and mention the missing benefit of a secreted heterologous toxin. Fusion proteins, consisting of secretion signal, toxin and heterologous antigen, are not at all described or suggested.
In above passages the prior art was presented, that describes bacterial carriers which express toxins heterologously and can be used as infection vaccines. In the cited examples mainly modifications regarding the expression or stability of toxins and their solubility were carried out, for instance insertion of strong expression promoter or fusion of a toxin to a secretion signal.
Other authors have also studied genetic fusions of toxins to heterologous antigens in live-vaccines. In these cases the toxin was mostly used as adjuvant. In some cases (e. g. (Brossier et al., 2000)) the heterologous antigen functioned as adjuvant and the toxin as proper antigen.
However, it is important to note, that in the mentioned cases, expression of the toxin-antigen gene fusion construct exclusively took place cytoplasmatically or periplasmatically. The toxin-antigen construct was neither fused to an additional secretion signal (which would lead to its complete secretion) nor was it directly secreted.
In the course of these toxin-antigen gene fusion constructs, recombinant E. coli (Clemens et al., 2004), Bacillus anthracis (Brossier et al., 2000), Shigella (Koprowski et al., 2000; Ranallo et al., 2005; Zheng et al., 2005) and Vibrio strains (Silva et al., 2003) were used. For Salmonella (summarized in (Garmory et al., 2002)), too, fusions of CT variants with antigens (Hajishengallis et al., 1996; Huang et al., 2000) or other toxins with antigens (Barry et al., 1996; Cardenas and Clements, 1993; Chabalgoity et al., 1997; Chabalgoity et al., 1996; Chabalgoity et al., 2000; Chabalgoity et al., 1995; Chacon et al., 1996; Jagusztyn-Krynicka et al., 1993; Khan et al., 1994a; Khan et al., 1994b; Lee et al., 2000; Pogonka et al., 2003; Schodel et al., 1990; Smerdou et al., 1996; Ward et al., 1999; Wu et al., 2000) have been described.
In the majority of these cases the main focus was on induction of a mucosal (antibody) immune response and for the induction of a systemic immune response, only subcutaneous, but not oral application was chosen [36].
Some work with Salmonella as supporter strain are limited to a mere characterization of the strain (Gomez-Duarte et al., 1995; Jagusztyn-Krynicka et al., 1993), others only analyze the mucosal and/or systemic antibody response and/or protection (Barry et al., 1996; Cardenas and Clements, 1993; Dunstan et al., 2003; Hajishengallis et al., 1996; Harokopakis et al., 1997; Khan et al., 1994a; Khan et al., 1994b; Pogonka et al., 2003; Smerdou et al., 1996; Somner et al., 1999). In all these cases, in which incidentally a fusion between antigen and tetanus toxin was used and which are exclusively used as infection vaccines, systemic cellular immune responses, particularly cytotoxic T-Cell responses, have not been studied.
Therefore, from an immunological point of view, no conclusions about a potential use as a tumor vaccine can be drawn from those studies, since their focuses were set on antibody mediated effects as infection vaccines.
Investigations that include an isotype analysis of the immune response studied appear to be more relevant. In fact, cellular immune responses are not directly measured in these cases, but the isotype profile of the antibody response allows a conclusion on the Th1/Th2 bias of the immune response. Antibody isotypes like IgG1 are associated with Th2 responses and isotypes like IgG2a are associated with Th1 responses. As already mentioned, Th1 responses are cellular dominated immune responses, whereas Th2 responses mainly represent humoral antibody-driven responses. Still, those studies do neither describe tumor vaccines nor do they suggest any use as anti-tumor agent.
One infection vaccine study based on Salmonella as carrier, which expresses a tetanus-toxin antigen fusion protein has been realized in dogs. The low antibody responses that were induced in dogs, show a Th1 bias, regarding the antibody profile, hence, a response that rather correlates with a cellular type immune response (Chabalgoity et al., 2000). Admittedly, dog immunology is only scarcely researched, and therefore it is not clear to which extent a dog's antibody profile can give information about a Th1 bias.
Studies in mice, performed by the same group, using comparable constructs, however showed an antibody profile with the same level for IgG1 as for IgG2a, which would indicate a mixed Th1/Th2 response.
Interestingly, an existing immunity against tetanus toxin, as it is found in most humans due to previous immunizations, causes another relatively strong induction of IgG 1, whereas IgG2a is hardly induced. This clearly indicates a distinct Th2 bias (Chabalgoity et al., 1995). For this reason, a tetanus toxin based live-vaccine as tumor vaccine is rather harmful for human use. For it can be expected, that a strong Th2 antibody-dominated response is induced in the majority of these patients, who show a tetanus toxin specific response.
Only few studies also analyze cellular immune response and compare genetic constructs with and without toxin fusion. In one case, for instance, a fusion of an antigen with and without tetanus toxin has been compared in Salmonella (Lee et al.). There, the tetanus toxin antigen fusion construct mainly increased the total antibody level, whereas the Th1/Th2 profile was hardly altered. Even the antigen specific CD4+ T cell secretion of typical Th1 cytokines, such as IFN-gamma and IL-2, respectively, only showed weak divergence. In an earlier study by the same group, cellular IFN-gamma levels had been measured, too. However, no comparison drawn with constructs without tetanus toxin (Chabalgoity et al.). Other studies with different gram-negative bacterial carriers, like Shigella (Koprowski et al.; Ranallo et al.; Zheng et al.) or Vibrio (Campos et al.; Ryan et al.), did not analyze isotypes and cellular immune responses, respectively, either.
In summary, it can be stated, that the studies mentioned clearly focus on the induction of an antibody-driven humoral immune response. Indeed, genetic toxin antigen constructs are used, but these are not provided with a secretion signal nor are they directly secreted. On no account, however, systemic cellular immune responses, in particular cytotoxic T-cell responses, were analyzed. What is more, such cellular cytotoxic T-cell responses cannot be concluded from humoral antibody response and cannot be detected if the antibody response is Th1/Th2-composed.
However, it is exactly these cellular cytotoxic T cell immune responses which are crucial for use in tumor vaccination therapy.
Hence, in terms of toxin-antigen fusions, which are restricted to mucosal infection vaccines only, the state-of-the-art does not allow any statement about possible use of any such constructs as tumor vaccines.
As already mentioned, expression of the genetic fusion constructs is realized without assistance of a secretion system. Toxins and toxin antigen constructs, respectively, are usually located cytoplasmatically as well as periplasmatically, i.e. between the two membranes. In order to induce an efficient cellular immune response the toxin must be freely accessible for the antigen-presenting cell (APC). Usually, native toxin is produced in the periplasm of gram-negative bacteria. This is sufficient for mere mucosal immune responses, because periplasmatic toxins can escape from the periplasm in the colon and consequently are accessible, too (Hunt and Hardy, 1991). This does not count, however, if the carrier targets antigen-presenting cells outside the colon, such as e.g. Peyer's Patches or lymphatic organs like lymph nodes or spleen.
In principle, two factors are crucial for the efficiency of a tumor vaccine: induction of a cellular immune response of the Th1-type and participation of components of the innate immune system, such as NK cells, NKT-cells and gamma-delta T cells, which play an important role for the efficiency of tumor therapy (Dunn et al., 2004).
The importance of these components of the innate immune systems lies on multiple levels. Properly activated NK- and gamma-delta T cells are able to locally produce large amounts of IFN-gamma. This interferon, which is also produced by specific Th1 polarized T-cells, has multiple functions, relevant to tumor therapy. One of its central functions is the inhibition of angiogenesis, which cuts off the tumor's oxygen and nutrients supply and de facto starves the tumor. Furthermore, NK cells possess receptors, which recognize MHC class I molecules. If these molecules are present on a cell, NK cells are inhibited.
As for a vaccine, which induces specific cytotoxic T-cells, tumor cells can be killed by these CTLs. If the tumor cell loses its capability to express MHC class I molecules, which occurs quite frequently in tumors, specific cytotoxic T-cells are ineffective. Hence, in this case, inhibition of NK cells is stopped, and they are able to eliminate tumor cells directly.
Consequently, it would be ideal if a tumor vaccine induces both components efficiently. There are contradictory data concerning the Th1-Th2 bias of a fused toxin adjuvant. As discussed earlier, toxin antigen fusion constructs applied in an isolated fashion obviously induce a strong Th2 polarized immune response. Some authors still describe a low Th2 bias for live-carriers; other authors see a slight Th1 bias.
However, these data again are exclusively based on non-secreted constructs. Induction of innate immunity by means of such kinds of infection vaccines has never been compared nor contemplated.
As already mentioned, the main reason is that the existing vaccines are mucosal infection vaccines, not tumor vaccines. Therefore, induction of Th1 immune responses, CTL immune responses and responses of the innate immune system were not in the focus. In contrast, regarding tumor vaccines, induction of these immune responses is indispensable.
Interestingly, in cell biology native toxins are commonly used as inhibitors of signaling pathways. So among others it has been shown, that native pertussis toxin, but not cholera toxin, is able to inhibit a particular apoptosis pattern of NK cells (Ramirez et al., 1994). A different research study was able to show that cholera toxin, but not its B-subunit, blocks specific NK cell functions (Poggi et al., 1996). Native pertussis toxin is used in order to inhibit chemotaxis of lymphocytes (Spangrude et al., 1985). Even if these studies do not deal with tumor vaccination, a person skilled in the art would conclude, that the use of toxins as tumor vaccines would be harmful, because the response of the innate immune system, crucial for tumor therapy, is rather inhibited than induced.
Other research studies were able to show that toxins like native pertussis toxin (but not inactive pertussis toxin) efficiently induce components of the innate immune system. Regarding immunotherapy against tumors, this would mean that, if at all, native toxins would need to be employed. However, due to toxicity reasons such kind of administration is infeasible. Further, such induction would inevitably lead to a Th2 directed secondary immune response, which in turn would be deleterious for tumor therapy (Boyd et al., 2005). Consequently, concerning the induction of an innate immune response, the prior art does not describe nor contemplates tumor vaccination. All the contrary, critical analysis of the literature even militates against the use of toxins in tumor therapy.
Interesting, though, is an analysis of the synergistic effect of toxins or their subunits with other stimulants, such as immune stimulating DNA oligonucleotides with hypomethylated CpG motives (CpG ODN) (Holmgren et al., 2005) or liposaccharides (LPS). In the case of LPS primarily the induction of monocytes appears to be increased mainly through the B-subunit of the toxins, whereas it is inhibited by the toxin as a whole (holotoxin) (Hajishengallis et al., 2004). However, these studies exclusively rely on the use of purified toxin-antigen-fusion constructs, to which substances like LPS or CpG are added as adjuvants. Furthermore, analysis there is only carried out on macrophages, which induce an adaptive immune response, but do not attack tumours directly. Hence, these studies have no significance regarding the induction of components of the innate immune system, in particular NK cells, which can attack tumors directly.
Other studies, however, show that NK cells can be activated and chemotacticly attracted by toxins like Pseudomonas aeruginosa Exotoxin A (Muhlen et al., 2004). Depending on the experimental system, Th1 responses can also be induced, although a suppression of NK cells and Th1 responses thereby mostly occurs (Michalkiewicz et al., 1999). However, these assays primarily aim at the analysis of the hepatotoxicity of Enterotoxin A and do not refer to tumor vaccination. Interestingly, the effects are highly dose-dependent, and only a slight change of dose can invert the effects. Yet, the authors could not reveal which response effectively occurs in vivo. As a consequence, the data do not provide a prediction which effects may occur if a toxin or even a detoxified toxin is used as adjuvant.
All in all, the immune response strongly depends on the particular system applied. In most cases, a mucosal anti-infection vaccine aims at the local manipulation of the immune system at the mucosa, in order to induce an efficient mucosal immune response (Lycke, 2005). However, these studies do not include the development of a tumor vaccine. In addition, those studies lack information about the induction of systemic cellular immune responses, particularly responses of cytotoxic T-cells, which are essential for tumor vaccination.
It has already been shown, that secretion of a heterologous antigen confers advantages for a systemic immune response (Hess et al., 1996). However, the secreted antigens described were not secreted toxin-antigen constructs; toxins or subunits thereof were not used. The thereby attainable immune responses in a transgenic tumor model were highly limited, too (Gentschev et al., 2005). Indeed, weak antibody and cytotoxic T-cell responses could be induced in this case, that partially protected from a tumor progression. However, not only the immune responses themselves, but the protection itself was limited. Likewise, these tumor vaccination studies lack a comparison with non-secreted constructs. Furthermore, the comparative studies were not carried out in the context of tumor vaccination (Hess et al., 1996) and are in contrast to further studies, that do not see any advantage with regard to secretion (Garmory et al., 2002; Roland et al., 2005).
In summary and as previously mentioned, the prior art is highly contradictory regarding secretion and, all in all, does not give any hint towards potential advantages of secreted toxin-antigen constructs in tumor therapy. All the contrary, critical analysis of the existing literature rather disagrees with such kind of use.
Bacterial toxins (Todar, 2002): At a chemical level, there are two types of bacterial toxins, lipopolysaccharides, which are associated with the cell walls of gram-negative bacteria, and proteins, which are released from bacterial cells and may act at tissue sites remote from the site of bacterial growth. The cell-associated lipopolysaccharide (LPS) toxins are referred to as endotoxins and the extracellular diffusible toxins are referred to as exotoxins.
Exotoxins are typically soluble proteins secreted by living bacteria during exponential growth but in some cases they are released by lysis of the bacterial cell. The production of the toxin is generally specific to a particular bacterial species that produces the disease associated with the toxin (e.g. only Clostridium tetani produces tetanus toxin; only Corynebacterium diphtheriae produces the diphtheria toxin). Both gram-positive and gram-negative bacteria produce soluble protein toxins.
In general there exist three classes of protein (exo-) toxins: (i) type I toxins (super-antigens), that bind to the host cell surface and modulate the immune response but are not translocated into the cell, (ii) type II toxins (pore-forming toxins), which act on the host cell membrane and make the host cell leak and die, and (iii) type III toxins (A-B toxins), which bind to the host cell via one specific receptor, are translocated into the cell, become active therein an modify proteins or other components of the host cell.
As indicated above, type III toxins, acting intracellularly with regard to host cells, consist of two components: one component (subunit A) is responsible for the enzymatic activity of the toxin; the other component (subunit B) is concerned with binding to a specific receptor on the host cell membrane and transferring the enzyme across the membrane. The enzymatic component is not active until it is released from the native (A+B) toxin. Isolated A subunits are enzymatically active but lack binding and cell entry capability. Isolated B subunits may bind to target cells (and even block the binding of the native toxin), but they are non-toxic.
There are a variety of ways that toxin subunits may be synthesized and arranged: A+B indicates that the toxin is synthesized and secreted as two separate protein subunits that interact at the target cell surface; A-B or A-5B or AB5 indicates that the A and B subunits are synthesized separately, but associated by noncovalent bonds during secretion and binding to their target; 5B or B5 indicates that the binding domain of the protein is composed of 5 identical subunits. AB or A/B denotes a toxin synthesized as a single polypeptide, divided into A and B domains, which may be separated by proteolytic cleavage. Examples of AB or A/B toxins are Diphtheria Toxin, Exotoxin A, Botulinum toxin and Tetanus Toxin. Examples of A-5B or AB5 toxins are Cholera Toxin and Shiga Toxin, whereas Anthrax Toxin LF and Anthrax Toxin EF are examples of A-B toxins.
Further relevant documents of the prior art comprise the following:
Michl et al. describe the use of bacteria and bacterial toxins as therapeutic agents for solid tumors. Toxin-Antigen fusion constructs are disclosed as well as bacterial targeting of such construct. Use of diphtheria toxin (DT), pseudomonas exotoxin A (PE) and clostridium perfringens enterotoxin (CPE) is studied. However, the authors do neither mention the use of cholera toxin nor do they show or render obvious secretion of bacteria-delivered toxin-antigen fusion constructs (Michl and Gress, 2004).
Lahiri gives an overview about different bacterial toxins and discuss their manifold uses. Although the author mentions toxin-antigen fusion proteins he is silent about cholera toxin and bacterial targeting of secreted toxin-antigen fusion constructs (Lahiri, 2000).
Lavelle et al. disclose molecules of infectious agents as immunomodulatory drugs. The authors also mention cholera toxin-antigen fusion proteins, but such constructs are only applied directly as proteins and not by means of genetically modified live vaccines (Lavelle et al., 2004).
WO 01/74383 is directed to chimeric antigen-enterotoxin mucosal immunogens and also mentions the use of cholera toxin subunits A2 and B. Such chimeric immunogens, however, comprises always A2 and B subunits at the same time and are intended for use in mucosal immunization but not in tumor therapy.
WO 02/077249 describes non-virulent Yersinia enterocolitica mutant strains for the delivery of heterologous proteins to specific mutated target cells. Use of cholera toxin subunit A1 is also mentioned but the patent document is silent about secretion and refers to the treatment of infections and infectious states only.
WO 2004/018630 discloses recombinant double stranded RNA phages encoding a double stranded eukaryotic expression cassette. Although cholera toxin subunit A is mentioned, the document is not of further relevance.
Holmgren et al. give a brief overview about the field of mucosal immunization and adjuvants. They discuss among others the effects of cholera toxin as mucosal adjuvants but do not disclose information about genetic expression systems or live vaccines and are also silent about tumor therapy (Holmgren et al., 2003).
Holmgren and Czerkinsky also give an overview about mucosal immunity and vaccines. However, this article is restricted to anti-infectives only and does not discuss or render obvious possible uses in the field of tumor therapy (Holmgren and Czerkinsky, 2005).
Another review by Freytag and Clements discusses mucosal adjuvants for application in anti-infective immunotherapy. Although cholera toxin is mentioned as mucosal adjuvants the authors are silent about secreted toxin-antigen constructs and tumor therapy as a possible field of administration (Freytag and Clements, 2005).
Shaw and Starnbach describe the use of modified bacterial toxins to deliver vaccine antigens. However, the article does not mention cholera toxin and further is limited to direct application of toxin-antigen fusion proteins for vaccination reasons (Shaw and Starnbach, 2003).
WO 03/072789 is directed to microorganisms as carriers of nucleotide sequences coding for cell antigens used for the treatment of tumors. Although the patent document mentions secretion and use in the field of tumor therapy it is silent about bacterial toxins and fusion proteins at all.
Gentschev, Dietrich and Goebel as well as Gentschev et al. describe bacterial targeting and its use in tumor vaccine development. However, these two documents do not mention the use of bacterial toxins and fusion proteins in tumor therapy (Gentschev et al., 2002a; Gentschev et al., 2002b).
WO 98/23763 discloses Vibrio cholerae cells expressing E. coli hemolysin B and D subunits along with a fusion polypeptide that includes a heterologous antigen fused to hylA. Further described is a Vibrio cholerae vaccine strain that expresses cholera toxin subunit B and a fusion polypeptide of a secretory signal sequence, heterologous antigen and choleratoxin A2 subunit. Last, a fusion polypeptide that includes cholera toxin B subunit fused to an antigenic portion of C. difficile toxin A or toxin B subunit is disclosed. However, the patent application does not mention use of a fusion protein of protein toxin plus a heterologous non-protein toxin antigen in tumor therapy.
Dietrich and co-workers discuss two vaccine delivery tools—haemolysin A and listeriolysin—that can be used for cell-mediated immunity. However, no protein toxin—heterologous antigen fusion proteins or co-expression is mentioned (Dietrich et al., 2003).
Gentschev et al. describe the use of the alpha-hemolysin secretion system of Escherichia coli for antigen delivery in the Salmonella typhi Ty21a vaccine strain. However, the authors do not mention the use of bacterial toxins and fusion proteins in tumor therapy (Gentschev et al., 2004).
WO 02/47727 is directed to therapeutic agents comprising a B-subunit of a protein toxin. The document only discloses fusion proteins of CtxB and EtxB with viral antigen. No bacterial vaccine or bacterial vaccine delivery is mentioned.
Cheng-hua S and co-workers describe a gene fusion of cholera toxin B subunit and HBV PreS2 epitope and the antigenicity of the fusion protein in direct immunization studies. However, no bacterial vaccine or bacterial vaccine delivery is mentioned (Cheng-hua et al., 1995).
Sanchez et al. disclose that cholera toxin B-subunit gene enhances mucosal immunoglobulin A, Th1-type and CD8+ cytotoxic responses when co-administered intradermally with a DNA vaccine (Sanchez et al., 2004). However, in this approach the authors use DNA as a carrier which acts as a Th1 promoting adjuvant on its own. In addition, the proteins are produced by host cells and not delivered directly or via abacterial carrier and are therefore directly available for eukaryotic cells.
WO 01/29233 is directed to chimeric immunogenic compositions and nucleic acids encoding them. However, no bacterial vaccine or bacterial vaccine delivery is mentioned.
WO 2007/044406 relates to methods for stimulating an immune response using a bacterial antigen delivery system that is based on a SopE bearing type III secretion signal. However, the patent application does not mention the use of bacterial toxins and fusion proteins in tumor therapy.
In summary, it can be concluded from the prior art that native toxins cannot be established for use in humans due to their strong toxicity. Further, their application in tumor therapy would be harmful because the response of the innate immune system, in particular that of NK cells, would be inhibited. However, it is this immune response which is a crucial component for a successful tumor therapy since tumor cells very frequently lose their capability to express MHC class I molecules and therefore are resistant to CTL recognition and attack.
Use of the detoxified toxin subunits, on the other hand, alone or fused to (heterologous) antigen proteins results in a strongly attenuated adjuvant effect an(/or even an induced systemic tolerance of the immune system as well as mucosally restricted antibody and Th2 type immune responses.
Furthermore, secretion of (heterologous) antigen-toxin fusion proteins, which is only described in the course of anti-infection vaccines (i.e. targeting the antigen or even the toxin itself), is said to not only display no advantages over cytoplasmatic expression, but to be generally rather unsuitable.
All in all, neither a tumor therapeutic approach is presented nor were a systemic induction of a Th1 dominated cellular immune response with IFN-gamma producing T-helper cells, an induction of CTL nor the activation of the innate immune system described or achieved, all of which are indispensable for tumor therapy.